In collaborative studies, the Auwerx and Bianco laboratories discovered that bile acids (BAs) increase energy expenditure in brown adipose tissue (BAT), preventing obesity and insulin resistance associated with a high fat diet2. This effect of Bas is critically dependent on induction of the cAMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) since it is lost in D2-/- mice. BA treatment of brown adipocytes and human skeletal myocytes increases D2 activity and oxygen consumption. These effects are independent of FXRa, and instead are mediated by increased cAMP production that stems from the binding of BAs with the G-protein-coupled receptor TGR5, which makes this GPCR an interesting target to modulate D2 activity. D2 activity can also be regulated through the D2-specific E3 ubiquitin ligase, WSB1, that inactivates D2 by ubiquitination7. Interestingly, BAT (in rodents) and skeletal muscle (in humans), the most thermogenically important tissues, both coexpress TGR5, D2 and WSB1, suggesting a tight regulation of D2- mediated T3 production in these tissues. The current proposal will test the hypothesis that TGR5 and WSB1 regulation of D2 activity and thyroid hormone activation is a key pathway involved in the control of energy homeostasis. Generating genetically engineered mouse models (GEMMs) with either germ line or spatially- and temporally-controlled TGR5 and WSB1 deficiency will allow us to test these hypotheses. The GEMMs that will result from this strategy will undergo extensive metabolic phenotypic characterization. These physiological data will be combined with in depth cellular and molecular profiling, enabling us: 1) to reconstruct the networks governed by TGR5-D2- WSB1 signaling;2) to determine the importance of localized thyroid hormone production in metabolic homeostasis;and 3) to validate modulation of TGR5-D2-WSB1 signaling as a strategy to prevent and combat metabolic disease. To achieve this we set forth to accomplish the following specific aims: 1. Development of GEMMs with germline and spatially- and temporally-controlled TGR5 and WSB1 mutations. 2. Tissue-specific TGR5 and WSB1 expression is critical in metabolic control. 3. Signaling through the TGR5-D2-WSB1 pathway affects cellular metabolic profile and gene expression profiles.